Age-related macular degeneration (AMD) is a complex disease caused by ageing, genetics, and the environment. While the genetics and environmental causes of AMD are being vigorously pursued, the exact role of ageing in AMD is neither well understood nor well studied. In other complex age-related diseases such as atherosclerosis and cancer, epigenetics is providing a basic understanding of how age contributes to disease development. Epigenetics is the study of long term covalent modification of the genome of somatic cells. These changes do not involve alterations in gene sequence and are therefore not mutations. Methylation of gene promoters at CG dinucleotide rich regions (CpG islands) is a major epigenetic modification leading to gene silencing. These changes occur in single cells as a stochastic process resulting in significant heterogeneity in populations of cells which are otherwise genetically identical. We hypothesize that genes functioning as suppressors of neurodegeneration, oxidative stress, or angiogenesis are silenced in the retinal pigment epithelium (RPE) as a function of age by epigenetic mechanisms. These changes are heterogeneous within the population of RPE cells leading to heterogeneous effects on the adjacent photoreceptors, Bruch's membrane, and choriocapillaris. To test this hypothesis, we will focus our initial studies on TIMP3, BRCA1, IGF2, and GSTP1. We have shown that the expression at the mRNA level of all four genes is downregulated with age in the RPE/choroid of the mouse. All four genes also have functions potentially related to AMD. TIMP3 is an angiogenesis inhibitor, BRCA1 and GSTP1 are suppressors of oxidative stress, and IGF2 is a trophic factor. Finally, the promoters of these genes all have CpG islands in the mouse and exhibit age-related methylation in a variety of human epithelial tissues. We will first quantify the age-related decline of expression for these genes on a single cell basis in the mouse RPE. Next we will investigate the age-related methylation of the promoters of these genes in whole mouse RPE/choroid. The successful completion of the these aims will establish epigenetics as an important new avenue for studying age-related changes in human RPE function as well as the technical ability to relate the epigenetic changes in individual RPE cells to localized areas of pathology. [unreadable] [unreadable] [unreadable]